Effect And Mechanism Of Ghrelin On Cerebral Microvascular Permeability Of Atherosclerotic Mice

Objective1.To study the changes of blood lipids,inflammatory factors and cerebral microvascular permeability in atherosclerotic model mice.2.Explore the effect of Ghrelin on blood lipids,inflammatory factors,and cerebral microvascular permeability in atherosclerotic mice and its possible mechanism.3.Explore the effect of Ghrelin on cerebral microvascular pericytes under ox-LDL culture and the regulatory mechanism of p38 MAPK-JNK signaling pathway in it.Methods1.Establish mouse models in the control group(Control),aortic atherosclerosis group(AS),and Ghrelin intervention group(Ghrelin).The control group mice were given ordinary food,the AS group and Ghrelin group were given high fat food for 12 weeks,the H&E and oil red staining of aortic root tissue sections were taken to verify that the mouse model was successfully constructed,then they were switched to normal food feeding,and the Ghrelin group mice were given intraperitoneal injection of Ghrelin for 4 weeks.2.Blood lipids and inflammatory factors of mice were detected by ELISA.The permeability of cerebral microvessels were detected by Evan blue and H&E staining.In addition,the ultrastructure of the cortical BBB was observed under the transmission electron microscope: the number of pericytes in the cerebral microvessels of the AS model mice decreased,and the pericytes swelled,and Ghrelin intervention can partially restore the number and morphology of pericytes.3.The distribution and number of pericytes in brain microvessels were detected by immunofluorescence.Western blot was used to detect the expression of PDGFR-? and p38 MAPK-JNK in the cerebral cortex of mice.4.Mouse primary brain microvascular pericytes were cultured in vitro and ox-LDL,Ghrelin or p38 MAPK-JNK modulator Hesperetin was added respectively.CCK-8 and western blot experiments was used to determine the optimal concentration of ox-LDL,Ghrelin and Hesperetin.ELISA and RT-q PCR was used to detect the relative expression of IL-6,MCP-1 and TNF-a in the culture media and within isolated pericytes,respectively.TUNEL was used to detect cell apoptosis.Western blot was used to detect the expression of p38 MAPK-JNK and Bcl-2/Bax/Cleaved Caspase-3.Results1.After 12 weeks of feeding,the mice in the Control group had no atherosclerosis,and the mice in the AS group had obvious atherosclerosis,indicating that the atherosclerosis model was successfully constructed;compared with the AS group mice,the serum ox-LDL and inflammatory factors IL-6,MCP-1,TNF-? levels were significantly down-regulated in the Ghrelin group mice.2.The Evans blue dye exuded from the BBB of AS mice was significantly more than that of Control mice.Compared with untreated AS mice,the Ghrelin treatment group had a significant decrease in BBB leakage of Evans blue.The immunofluorescence showed that the number of pericytes(PDGFR-? positive)in the brain microvessels(CD31 positive)of AS model mice decreased,while Ghrelin intervention could partially restore the number of pericytes.In addition,the ultrastructure of BBB in cortex was observed under transmission electron microscope.The pericytes was swollen in AS mice,while in Ghrelin mice,the morphology of pericytes partially restored.It was also observed that the protein of PDGFR-?(pericytes markers)in the brain tissue of AS mice reduced,but the expression of PDGFR-? up-regulated in Ghrelin mice.3.The levels of inflammatory factors such as IL-6,MCP-1 and TNF-?in pericytes in the ox-LDL group were significantly increased,Ghrelin intervention significantly lowered the levels of IL-6,MCP-1,and TNF-?in proteins and transcript levels.Tunel experiment showed that pericyte apoptosis increased in the ox-LDL group,and Ghrelin intervention could reduce pericyte apoptosis.Western-blot test results indicated that Ghrelin intervention could down-regulate the expression of p38 MAPK-JNK,Bax/Cleaved Caspase-3 and up-regulate the expression of Bcl-2 in pericytes.Hesperetin(p38 MAPK-JNK agonist)partially reversed the inhibitory effect of Ghrelin on pericyte secreting of inflammatory factors and apoptosis,as evidenced by the elevated levels of Bax/Cleaved Caspase-3 and the decreased expression of Bcl-2.Conclusions1.Excessive fat and inflammation,associated with atherosclerosis,can promote cerebral microvascular leakage.2.Ghrelin,attenuated the damaging effects of atherosclerosis,improved cerebral microvascular leakage,lowered serum inflammatory factors,and restored pericyte morphology and number.3.Ghrelin can significantly down-regulate the levels of inflammatory factors and reduce the apoptosis of pericytes stimulated by ox-LDL.The underlying mechanism involved the downregulation of the p38MAPK-JNK pathway in pericytes.